Identification of putative virulence-associated genes of Streptococcus pyogenes by genomic subtractive hybridization

The fragments of virulence genes in Streptococcus pyogenes were isolated with suppression subtractive hybridization (SSH). Then these fragments were directly inserted into T/A cloning vector to set up subtractive library, and the amplification of the library was carried out after transferring into Escherichia coli TOP10. Dot blot was used to screen the subtracted library, and the differentially expressed cDNA fragments were sequenced and analyzed with basic local alignment search tool (BLAST) search. As a result, a smear with dots ranging from 100 to 2000 bp was obtained. Partial positive clones in the library were randomly selected and successfully sequenced. Five-twelfth (5/12) of sequences showed no homology and presumably represented unknown genes, and 7/12 had a high similarity to the known genes. In a conclusion, virulence genes subtracted library of S. pyogenes is constructed successfully with SSH and T/A cloning techniques. The library is efficient and lays solid foundation for screening and cloning new and specific virulence genes of S. pyogenes.   Key words: Streptococcus pyogenes; suppression subtractive hybridization; virulence gene; dot blot.